Bionanomechanics with Optical Tweezers: Molecular Machines under Tension Erik Schäffer Center for Plant Molecular Biology (ZMBP) University of Tübingen, Germany www.zmbp.uni-tuebingen.de/nano 16 April 215 Cellular Nanoscience 1/21 Mechanical Processes Fulfill Essential Cellular Functions Gary Banker 2 Cell division (Ted Salmon 26) William M. Saxton 28 Transport in neurons Single-molecule force measurements 2/21
Optical Tweezers: Ideal Tool for Motor Protein Mechanics detector focused laser Kinesin-1 (BioVisions, Harvard University) microsphere motor protein microtubule surface 3/21 Outline Optical Tweezers A Sensitive Position & Force Transducer Bionanomechanics Transport & Friction of Molecular Machines DNA repair: Molecular Clamping Outlook 4/21
Invisible, High-Power, Infrared Lasers for Trapping Biological matter absorbs little in the near infrared 5/21 Microsphere Remains Trapped Against Hydrodynamic Flow 6/21
Contact-Free, Live Cell Mechanical Manipulation trapped bacteria, Block laboratory 1989 7/21 Optical Tweezers for High-Resolution 3D Measurements beam dump QPD intensity feedback Nd:YVO 4 laser (5W @ 164 nm, linearly polarized) heat sink QPD intensity monitor polarizer λ/2 Faraday isolator LED iris servo servo QPD position detection xyz piezo inertial stage (8 x 8 x 3.5 mm 3 ) piezo translation stage (3 x 3 x 1 µm 3 ) temperaturecontrolled objectives stabilized to within.1 C IR piezo tilt mirror (xy, 5 mrad) shutter VIS video camera adjustable zoom (.4-2.x) IR block Langmuir 27, J. Microsc. 27, Opt. Express 28, 29, 211 8/21
Molecular Position & Force Resolution surface optical gradient fixed microsphere Position (nm).6.4.2..2.4 1 2 3 4 5 6 7 Time (s) Å, fn, µs resolution with.1 C stability Opt. Express 29 9/21 Antireflection-Coated Microspheres Improve Optical Trapping Stabilizing gradient force Destabilizing scattering force Reduce scattering force by coating partially antireflection-coated glass Science 1999 1/21
Nanonewton Optical Forces with Coated Microspheres silica silica-coated polystyrene 1 µm surface x z y polystyrene titania Antireflection coating reduces scattering via destructive interference TEM.5 µm High-refractive index titania particles: 1 nn force with optimized tweezers Novel experiments feasible Opt. Express 28, Patent Appl. 28, Langmuir 211, Phys. Rev. Lett. 211, Nature Photon. 212 11/21 Kinesin Motors Take 8 nm Steps 2 Position (nm) 15 1 2 µm (5 real time) 5 LED-DIC J. Microsc. 27 1. 1.5 2. 2.5 3. Time (s) 12/21
Kinesin-1 Steps Efficiently & Transports Piconewton Loads 16 12 127 nm/s at 1 µm ATP 5 4 Position (nm) 8 4 efficiency = (max. load step size) / ATP energy (5 pn 8 nm)/1 pn nm = 4% 3 2 1 Force (pn) macroscopic engines: typically 15 35% 26 28 3 32 34 Time (s) 13/21-1 Kinesin-8 Is a Weak Motor That Depolymerizes Microtubules & Slips microtubule kinesin-8 + 4 32 (5 real time) Position (nm) 24 16 Slippage for F > 1 pn (Biophys. J. 213) 1 min 1 µm Force (pn) 8 2 1 54 55 56 Time (s) Friction? (Science 29) 14/21
Friction Is Related to Diffusion Friction resists the relative motion of two bodies in contact. Contact mediated by adhesive bonds between molecules Friction: force necessary to deform & break these bonds Einstein relation: Kinesin-8 diffuses in ADP γ = k BT D 2 µm, 17 real time Kinesin-8: Model System for Protein Friction 15/21 Friction Limits Diffusive & Directed Motor Movement 5 4 + MSD x 2 (µm 2 ).15.1.5 2 µm 1 s. 2 4 6 8 1 12 Time t (s) Diffusion coefficient D from single molecule tracking Friction force F friction (pn) 3 2 1 1 2 3 2 4 8 12 µm/s 4 5.2.4.6.8 Stage position (µm) Frictional drag coefficient γ from optical tweezers dragging Einstein relation holds for single molecular machines (Science 29) 16/21
Motivation Optical Tweezers Molecular Machines Summary Mechanics of DNA Repair: Homologous Recombination double-strand break repair Rad51 Rad52 RecA Redβ strand invasion single strand annealing homologs Z 17/21 Motivation Optical Tweezers Molecular Machines MBP Summary Redβ Forms Complexes with DNA 1 nm Redβ catalyzes DNA strand annealing without ATP Redβ forms helical superstructures with DNA Redβ is related to Rad52 Passy et al., PNAS 96, 4279 (1999) Rad52 ring Erler et al., J. Mol. Biol. 391, 586 (29) only Redβ Redβ-DNA complex 18/21 Z MBP
Redβ Strongly Clamps DNA Strands Together 2 pull Force (pn) 15 1 5 Redβ 123 b 11 12 Redβ Force (pn) 18 17 1..5 Stage position (µm) 16 18 2 22. 24 1 2 Time (s) 3 Time (s) 19/21 Mechanical Insight into Chromosome Segregation microtubules spindle poles Diffusive contacts: 1. Rapid microtubule end targeting (MCAK, XMAP215) kinetochore chromosome cross-linker 2. Chromosome attachment to shrinking microtubule tips (Dam1, Ndc8) 3. Cross-linking of microtubules while allowing for relative sliding (Eg-5, Ase1p, Ncd) 4. Processivity enhancement via slip state (load sharing?) (Kinesin-8, Eg-5) 2/21
Motivation Optical Tweezers Molecular Machines Summary Acknowledgements The Team: Mohammed Mahamdeh Volker Bormuth Anita Jannasch Marcel Ander Collaborators: Funding: Joe Howard (Yale) Francis Stewart (BIOTEC, TU Dresden) Alfons van Blaaderen (Utrecht) Emmy Noether-Program, DFG 21 ERC Starting Grant 21/21 Z MBP